The purpose of this project is to understand the molecular mechanisms responsible for replication of picornaviruses in susceptible target cells. This virus family includes numerous human pathogens (poliovirus, coxsackievirus, echovirus, enteroviruses, rhinoviruses, hepatitis A virus). Infection of cells with these viruses leads to major changes in the host cell's structure and metabolic activity. Cellular protein and RNA synthesis are inhibited; the intracellular membrane network becomes rearranged into vesicles that surround and provide a scaffold for viral RNA replication complexes; cellular proteins are subverted into facilitating viral protein and RNA synthesis. The unique combination of viral and cellular proteins together accomplish a highly efficient production of viral RNA, proteins, and particles. We are studying the activities of individual viral gene products, expressed alone or in combination, in cultured human cells, to determine their specific roles in the replication process. Assays for individual steps in the reaction have been developed and are being characterized biochemically. Mutations in individual viral proteins have been engineered and analyzed for their effects on different steps in the replication process. We have discovered that synthesis of specific viral protein(s) induce the activation and recruitment to membranes of a group of cellular proteins that play specific roles in membrane trafficking, vesicle formation, and morphology, which collectively results in the formation of viral RNA replication complexes in infected cells. These cellular proteins appear to trigger the formation of a membranous scaffold for viral RNA synthesis and perhaps recruit additional proteins that are required for virus replication. Understanding the precise biochemical activities of viral proteins in the replication process will allow the development of new strategies for vaccine development and the design of antiviral drugs.